Endotoxemia As Possible Cause of Inflammatory Bowel Diseases-Associated Anemia

Inflammatory bowel diseases (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions of the gastrointestinal tract and highly prevalent in Australia and USA. Anemia is debilitating and a common complication of IBD associated with chronic fatigue, poor quality of life and increased rate of hospitalization. However, current treatments for anemia in IBD are limited to iron supplementation which has limited efficacy. In this study, we investigated the cause of anemia in IBD patients and in a mouse colitis model. We have previously shown that endotoxins/lipopolysaccharides (LPS) inhibit bone marrow (BM) erythropoiesis in a TLR4- and MyD88-dependent manner in mice. Therefore, we first measured blood plasma concentrations of endotoxins in IBD patients and mouse model of acute colitis. Blood endotoxin concentrations were significantly increased relative to healthy controls in IBD patients (~4-fold in UC and ~2.2-fold in CD patients) and in mice with colitis induced by sodium dextran sulphate (DSS) (~4-fold). This is consistent with disruption of the intestinal barrier observed in IBD patients and DSS-treated mice allowing translocation of endotoxins produced by the gut bacterial flora into the circulation. A positive correlation between anemia and endotoxemia was found in IBD patients. To investigate the effect of gut inflammation on erythropoiesis, we measured the markers of erythropoiesis in mice with acute colitis induced by 3% DSS in drinking water. Mice with acute colitis had significant anemia with reduced number of red blood cells (RBCs; controls mean 9.0 x 10¹² ± 0.7 /L vs DSS-treated 7.5 x 10¹²± 1.1 /L), hemoglobin (HgB; mean 140 ± 9.1 g/L vs 117 g/L ± 15 g/L) and hematocrit (HCT; mean 0.41 ± 0.02 vs 0.34 ± 0.05) compared to control mice. In line with this, we found that acute colitis in mice caused a marked whitening of the bone marrow (BM) with reduced number of erythrocytes (mean 4.3 x 10⁶ ± 1.8 erythrocytes/femur vs 2.3 x 10⁶ ± 1.4 erythrocytes/femur), accompanied by shortened RBC half-life (t^1/2 = 19.7 ± 2.9 days in controls vs t^1/2 = 12.8 ± 4.4 days in DSS treated mice) in the blood and increased blood concentration of interferon-γ (mean 4.7 pg/mL ± 0.8 vs 7.6 pg/mL ± 2.7). Interferon- γ is known to reduce RBC lifespan by increasing their clearance in the spleen. Acute colitis increased RBC size (~1.15-fold increase in red cell width and ~ 3.5-fold increase in blood reticulocytes) consistent with colitis induced anemia. This reduced medullary erythropoiesis was compensated in part by extramedullary erythropoiesis in the spleen. Patients with IBD had increased levels of hepcidin (~4-fold increase) and interleukin -6 (IL-6; ~1.3-fold increase) in their blood. However, mice with acute colitis only showed an increase in blood IL-6 concentration (~9-fold increase) but no change in hepcidin, suggesting that there is a combination of both iron-deficiency anemia and anemia of inflammation in these settings.

We have previously shown that endotoxin inhibits erythropoiesis indirectly by deregulating erythroblastic islands macrophages (EBI Mφ) which express the CD169/Siglec1 antigen and endotoxin receptor TLR4. To investigate the role of EBI Mφ on colitis-induced anemia, we specifically deleted TLR-4 in CD169⁺ Mφ by crossing Tlr4^flox/flox mice with Siglec1Cre mice (expressing Cre recombinase in CD169⁺ tissue Mφ). Mice lacking Tlr4 gene specifically in CD169⁺ Mφ did not develop anemia in response to DSS with normal blood RBC, HgB, HCT, reticulocytes numbers, BM erythrocyte numbers and no extramedullary erythropoiesis in the spleen compared to mice with Tlr4 gene in CD169+ macrophages. In addition to the protective effect of Tlr4 deletion in CD169+ Mφ on anemia, we found that these mice were also protected from colitis with improved body weight, colon length and histological scores suggesting that endotoxemia may contribute to both anemia and colitis in IBD. In conclusion, we show for the first time that inflammation via endotoxins may play a crucial role in anemia in IBD and there is a correlation between anemia and endotoxemia in IBD.

An:AbbVie,Bristol Myers Squibb, Eli Lilly, Dr Falk, Ferring, Glaxo-Smith Kline, Janssen, Pfizer, Samsung, Sandoz, Shire, and Takeda Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.